Aptamer-free upconversion nanoparticle/silk biosensor system for low-cost and highly sensitive detection of antibiotic residues.

The detection of antibiotics is crucial for safeguarding the environment, ensuring food safety, and promoting human health. However, developing a rapid, convenient, low-cost, and sensitive method for antibiotic detection presents significant challenges. Herein, an aptamer-free biosensor was successfully constructed using upconversion nanoparticles (UCNPs) coated with silk fibroin (SF), based on Förster resonance energy transfer (FRET) and the charge-transfer effect, for detecting roxithromycin (RXM). A synergistic FRET efficiency was achieved by utilizing alizarin red and RXM complexes as energy acceptors, with UCNP as the energy donor, and immobilizing an ultrathin SF protein corona within 10 nm. The biosensor detects RXM in deionized water with high sensitivity primarily through monolayer adsorption, with a detection range of 1.0 nM-141.6 nM and a detection limit as low as 0.68 nM. The performance of this biosensor was compared with the ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for detecting antibiotics in river water separately and a strong correlation between the two methods was observed. The biosensor exhibited long-term stability in aqueous solutions (up to 60 d) with no attenuation of fluorescence intensity. Furthermore, the biosensor's applicability extended to the highly sensitive detection of other antibiotics, such as azithromycin. This study introduces a low-cost, eco-friendly, and highly sensitive method for antibiotic detection, with broad potential for future applications in environmental, healthcare, and food-related fields.

3D Bioprinted Human Skin Model Recapitulating Native-Like Tissue Maturation and Immunocompetence as an Advanced Platform for Skin Sensitization Assessment.

Physiologically-relevant in vitro skin models hold the utmost importance for efficacy assessments of pharmaceutical and cosmeceutical formulations, offering valuable alternatives to animal testing. Here, an advanced immunocompetent 3D bioprinted human skin model is presented to assess skin sensitization. Initially, a photopolymerizable bioink is formulated using silk fibroin methacrylate, gelatin methacrylate, and photoactivated human platelet releasate. The developed bioink shows desirable physicochemical and rheological attributes for microextrusion bioprinting. The tunable physical and mechanical properties of bioink are modulated through variable photocuring time for optimization. Thereafter, the bioink is utilized to 3D bioprint "sandwich type" skin construct where an artificial basement membrane supports a biomimetic epidermal layer on one side and a printed pre-vascularized dermal layer on the other side within a transwell system. The printed construct is further cultured in the air-liquid interface for maturation. Immunofluorescence staining demonstrated a differentiated keratinocyte layer and dermal extracellular matrix (ECM)-remodeling by fibroblasts and endothelial cells. The biochemical estimations and gene-expression analysis validate the maturation of the printed model. The incorporation of macrophages further enhances the physiological relevance of the model. This model effectively classifies skin irritative and non-irritative substances, thus establishing itself as a suitable pre-clinical screening platform for sensitization tests.

Silk fibroin nanoparticles and ß-tricalcium phosphate loaded tissue engineered gelatin bone scaffolds: A Nature-based, low-cost solution.

Tissue engineering has recently attracted attention as an alternative to traditional treatment methods for tissue and organ damage. Since bone is one of the most important vital parts of the body, the treatment of bone damage is important. Silk fibroin is a natural polymer with properties such as biocompatibility and biodegradability, which attracts attention with its controlled release, especially in drug delivery systems. In this study, gelatin-based scaffolds loaded with silk fibroin nanoparticles and ß -tricalcium phosphate (ß -TCP) were developed to be used as a potential drug delivery system in bone tissue engineering. The chosen nanoparticle formulation has a 294 nm average diameter with a 0.380 polidispersity index (PDI). In vitro characterization of scaffolds was performed by mechanical, morphological characterization, swelling capacity, Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared Spectroscopy (FT-IR) measurements, and biocompatibility was evaluated by cell culture studies. Swelling index, tensile strength, elongation at break, and Young modulus of the ß -TCP and silk nanoparticles loaded scaffold were found as 456%, 1.476 MPa, 6.75%, and 24 MPa, respectively. In vitro cell culture studies have shown that scaffolds prepared in the present study can accelerate osteoblast differentiation and increase the healing rate of bone tissues. In addition, they have the potential to be used as a drug delivery system in bone tissue engineering that needs to be evaluated with further studies.

Silk Fibroin-Based Piezoelectric Sensor with Carbon Nanofibers for Wearable Health Monitoring Applications.

The continuous real-time monitoring of human health using biomedical sensing devices has recently become a promising approach to the realization of distant health monitoring. In this paper, the piezoelectric characteristics of the silk fibroin (SF) natural polymer were analyzed as the material used for obtaining sensing information in the application of distance health monitoring. To enhance the SF piezoelectricity, this paper presents the development of a novel SF-based sensor realized by combining SF with different carbon nanofiber (CNF) densities, and for such newly developed SF-based sensors comprehensive performance analyses have been performed. Versatile methods including the scanning electron microscope, Fourier transform infrared spectroscopy, Raman and X-ray diffraction measurements and impedance analysis were used to study the morphologic, mechanical and electrical properties of the developed SF-based sensor. The SF with CNF samples was analyzed for three different pressure loads (40 N, 60 N and 80 N) in 500 compression test cycles. The analyses thoroughly describe how combining natural polymer SF with different CNF densities impacts the piezoelectricity and mechanical strength of the proposed SF-based sensor. The developed piezoelectric SF-based sensors were further tested on humans in real medical applications to detect generated piezoelectric voltage in versatile body movements. The maximum piezoelectricity equal to 2.95 ± 0.03 V was achieved for the jumping movement, and the SF sample with a CNF density equal to 0.4% was tested. Obtained results also show that the proposed SF-based sensor has an appropriate piezoelectric sensitivity for each of the analyzed body movement types, and that the proposed SF-based sensor can be applied in real medical applications as a biomedical sensing device. The proposed SF-based sensor's practical implementation is further confirmed by the results of cytotoxicity analyses, which show that the developed sensor has a non-toxic and biocompatible nature and can be efficiently used in skin contact for biomedical wearable health monitoring applications.

Formulation, characterization, and cellular toxicity assessment of tamoxifen-loaded silk fibroin nanoparticles in breast cancer.

Silk fibroin (SF) is a natural polymeric biomaterial that is widely adopted for the preparation of drug delivery systems. Herein, we aimed to fabricate and characterize SF nanoparticles loaded with the selective estrogen receptor modulator; tamoxifen citrate (TC-SF-NPs) and to assess their in vitro efficacy against breast cancer cell lines (MCF-7 and MDA-MB-231). TC-loaded SF-NPs were characterized for particle size, morphology, entrapment efficiency, and release profile. In addition, we examined the in vitro cytotoxicity of TC-SF-NPs against human breast cancer cell lines and evaluated the anticancer potential of TC-SF-NPs through apoptosis assay and cell cycle analysis. Drug-loaded SF-NPs showed an average particle size of 186.1 ± 5.9 nm and entrapment efficiency of 79.08%. Scanning electron microscopy (SEM) showed the nanoparticles had a spherical morphology with smooth surface. Tamoxifen release from SF-NPs exhibited a biphasic release profile with an initial burst release within the first 6 h and sustained release for 48 h. TC-SF-NPs exerted a dose-dependent cytotoxic effect against breast cancer cell lines. In addition, flow cytometry analysis revealed that cells accumulate in G0/G1 phase, with a concomitant reduction of S- and G2-M-phase cells upon treatment with TC-SF-NPs. Consequently, the potent anticancer activities of TC-SF-NPs against breast cancer cells were mainly attributed to the induction of apoptosis and cell cycle arrest. Our results indicate that SF nanoparticles may represent an attractive nontoxic nanocarrier for the delivery of anticancer drugs.

Synthesis and assessment of sodium alginate-modified silk fibroin microspheres as potential hepatic arterial embolization agent.

Transcatheter arterial chemoembolization (TACE) is well known as an effective treatment for hepatocellular carcinoma (HCC). In the present study, a novel embolic agent of sodium alginate (SA)-modified silk fibroin (SF) microspheres was successfully prepared by emulsifying cross-linking method. The SA-modified SF microspheres were evaluated for the ability of embolization by investigating the morphology, particle size, swelling ratio, degradation, cytotoxicity, blood compatibility, and in vivo embolization. The results found that SA-modified SF microspheres had smooth surfaces and good sphericity. Swelling ratio of the microspheres can meet the requirements of arterial embolic agent and have pH and temperature sensitivity. Furthermore, hemolytic and anticoagulant studies have proved that the microspheres have good blood compatibility. Cytotoxicity tests indicated that the microspheres could promote the proliferation of fibroblasts and HUVEC. In vivo embolization evaluation of microspheres revealed that the arteries could be embolized by SA-modified SF microspheres in 3 weeks. The ability of drug loading and releasing of microspheres was proved by the controlled release profile of Adriamycin hydrochloride. The findings indicated that the SA-modified SF microspheres can be used as a potentially biodegradable arterial embolic agent for liver cancer therapy.

Structural and functional evaluation of oxygenating keratin/silk fibroin scaffold and initial assessment of their potential for urethral tissue engineering.

In this study, we report a new type of oxygen-generating scaffold, composed of human keratin, silk, gelatin and calcium peroxide (CPO). After mixing the silk/keratin (60:40) with 2% gelatin and 20% CPO, the film demonstrated excellent mechanical properties, non-cytotoxicity and oxygen-generative ability. The detailed structure of scaffold was revealed by confocal laser and electronic scanning microscopy. The gelatin formed the network structure, which mixed with silk fibroin and keratin. The CPOs were embedded into scaffold. A shell-core structure was formed in the CPO particles, in which the CPO was located in the core and the gelatin was mainly wrapped around the CPO. Furthermore, the oxygen-release test showed that scaffold was able to steadily release high level of oxygen over two weeks in vitro. In addition, the anti-bacterial function was also proved in the scaffold. Films with CPO enhanced the repair in dog urethral defect models, resulting in patent urethra. Improved organized muscle bundles and epithelial layer were observed in animals treated with CPO films compared with those treated with non-CPO films. This study suggests that this biomaterial could be suitable for tissue engineered urinary tract reconstruction.

Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation.

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial (HCE) cells. Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell-attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favour the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

A silk fibroin based hepatocarcinoma model and the assessment of the drug response in hyaluronan-binding protein 1 overexpressed HepG2 cells.

Microenvironment around tumor cells plays an important role in its malignancy or invasiveness. Hyaluronan (HA), a major component of extracellular matrix is found to be elevated in most of cancerous niche/microenvironment and performs regulatory role in the progression of tumors and metastasis. Overexpression of the hyaladherin, hyaluronan-binding protein 1 (HABP1) in the hepatocarcinoma cells (HepG2) termed as HepR21 leads to enhanced cell proliferation with increased HA 'pool' associated with HA 'cables' indicating elevated tumorous potential under 2D culture conditions. For in vitro experimentation, scaffold based three dimensional niche modeling may have greater acceptance than conventional 2D culture condition. Thus, we have examined the influence of intrinsic properties of non-mulberry tropical tasar silk fibroin on the HepR21 cells in order to develop a 3D hepatocarcinoma construction to act as model. The scaffold of tasar silk fibroin of Antheraea mylitta when efficiently loaded with transformed hepatocarcinoma cells, HepR21; exhibits enhanced adhesiveness, viability, metabolic activity, proliferation and enlarged cellular morphology in 3D compared to its parent cell line HepG2, supporting the earlier observation made in 2D system. In addition, formation of multicellular aggregates, the indicator of tumor progression is also revealed in silk based 3D culture conditions. Further, the use of 4-MU (a hyaluronan synthase inhibitor) on HepR21 cells reduces the HA level and downregulates the expression of growth promoting factors like pAKT and PKC; while upregulating the expression of the tumor suppressor p53. Thus, 4-MU efficiently reduces the tumor potency associated with increased HA pool as well as HA cables and the effect of 4-MU doubling up as an anticancer agent in 2D and 3D are also comparable. The in vitro 3D multicellular model demonstrates the insight of hepatocarcinoma progression and offers the predictability of cellular response to transfection efficacy, drug treatment and therapeutic intervention.